Comparison of the effects of the PI3K/mTOR inhibitors NVP-BEZ235 and GSK2126458 on tamoxifen-resistant breast cancer cells

Abstract

Background: Treatment with anti-estrogens or aromatase inhibitors is commonly used for patients with estrogen receptor-positive (ER+) breast cancers; however resistant disease develops almost inevitably, requiring a choice of secondary therapy. One possibility is to use inhibitors of the PI3K/mTOR pathway and several candidate drugs are in development. We examined the in vitro effects of two inhibitors of the PI3K/mTOR pathway on resistant MCF-7 cells.

Methods: We cultured MCF-7 cells for prolonged periods either in the presence of the anti-estrogen tamoxifen (3 sub-lines) or in estrogen free medium (2 sub-lines) to mimic the effects of clinical treatment. We then analyzed the effects of two dual PI3K/mTOR phosphoinositide-3-kinase inhibitors, NVP-BEZ235 and GSK2126458, on the growth and signaling pathways of these MCF-7 sub-lines. The functional status of the PI3K, mTOR and ERK pathways was analyzed by measuring phosphorylation of AKT, p70S6K, rpS6 and ERK.

Results: The derived sub-lines showed increased resistance to tamoxifen but none exhibited concomitantly increased sensitivity to the PI3K inhibitors. NVP-BEZ235 and GSK2126458 acted mainly by induction of cell cycle arrest, particularly in G1-phase, rather than by induction of apoptosis. The lines varied considerably in their utilization of the AKT, p70S6K and ERK pathways. NVP-BEZ235 and GSK2126458 inhibited AKT signaling but NVP-BEZ235 showed greater effects than GSK2126458 on p70S6K and rpS6 signaling with effects resembling those of rapamycin.

Conclusion: Increased resistance to tamoxifen in these MCF-7 sub-lines is not associated with hypersensitivity to PI3K inhibitors. While both drugs inhibited AKT signaling, NVP-BEZ235 resembled rapamycin in inhibiting the mTOR pathway.

Figures

Figure 1

Effects of BEZ235 and GSK212 in MCF-7 parental and its derived sub-lines in proliferation and apoptosis. MCF-7 parental and its sub-lines were exposed to indicated concentration of BEZ235 and GSK212 (A) for 3 days, and cell proliferation was measured by sulforhodamine B assay. Bars represent percent changes in cell density after 72 h compared with initial amount present at the treatment start and expressed as the mean ± standard error from three experiments. Immunoblotting for cleaved PARP (cPARP) in MCF-7 cell lines treated with different concentration of BEZ235 (B) or GSK212 (C) for 72 h. Actin was used as a loading control. Bands were normalized to total protein and bars represent changes in fold compared with untreated cells and expressed as the mean ± standard deviation from three experiments. Representative blots are shown above bar graph. *Significant difference from treatment control (p

Figure 2

G1/S cell cycle arrest in MCF-7 cell lines treated with indicated concentration of BEZ235 (A) or GSK212 (B) for 24 h analyzed by flow cytometry. Results were shown as the mean ± standard deviation from two experiments. *Significant difference from treatment control (p < 0.05).

Figure 3

Immunoblotting for PI3K pathway signaling proteins in MCF-7 cell lines treated with different concentrations of BEZ235 or GSK212 for 24 h. BEZ235 and GSK212 inhibit signaling through the PI3K/mTOR pathway and its downstream effectors Akt (A), p70S6K (B), rpS6 (C) irrespective of the cellular growth response to the inhibitors. Effect on ERK activation was assessed (D). Immunoblotting used antibodies for p-Akt(S473), p-p70S6K(T389), p-rpS6 (S235/236), p-ERK(T202/Y204) and their respective proteins (which were used as loading controls). Bands were normalized to total protein and bars represent changes in fold compared with untreated cells and expressed as the mean ± standard deviation from three experiments. Representative blots are shown above bar graph. *Significant difference from treatment control (p

Figure 3

Immunoblotting for PI3K pathway signaling proteins in MCF-7 cell lines treated with different concentrations of BEZ235 or GSK212 for 24 h. BEZ235 and GSK212 inhibit signaling through the PI3K/mTOR pathway and its downstream effectors Akt (A), p70S6K (B), rpS6 (C) irrespective of the cellular growth response to the inhibitors. Effect on ERK activation was assessed (D). Immunoblotting used antibodies for p-Akt(S473), p-p70S6K(T389), p-rpS6 (S235/236), p-ERK(T202/Y204) and their respective proteins (which were used as loading controls). Bands were normalized to total protein and bars represent changes in fold compared with untreated cells and expressed as the mean ± standard deviation from three experiments. Representative blots are shown above bar graph. *Significant difference from treatment control (p

Figure 4

Immunoblotting for p-Akt(S473) and ER antibodies. MCF-7 sub-lines were treated with the indicated concentrations of BEZ235 or GSK212 overnight. Actin is shown as a loading control. Bands were normalized to actin and bars represent changes in fold compared with untreated cells and expressed as the mean ± standard deviation from three experiments. Representative blots are shown above bar graph. *Significant difference from treatment control (p